Effect And Mechanisms Of FOXO1on Osteoclastogenesis And Its Function

Implant denture is regarded as "the third set of teeth" and has more irreplaceableadvantages than traditional dental prosthesis. However, relevant risks and diseases such asimplant failure and peri-implantitis also turn up with dental implants being more and morepopular, in which latter is the common symptom. Peri-implantitis refers to inflammation oftissues around dental implants, which includes reversible implant mucositis involving softtissue and peri-implantitis involving surpporting bone. If not treated in time, there will becontinuous bone resorption and the integrate of implant-bone surface will be destroyed, whichleads to implant failure. Osteoclasts are the unique multinucleated cells in vivo that isresponsible for resorbing bone. They are generated from myeloid progenitors and have thespecial phenotype. Osteoclasts degrade bone mineral substance quickly by secretingcollagenase and other hydrolytic enzyme, which then results in bone resorption. In physicalcondition, the dynamically balance of bone tissue is controlled by both bone degradation byosteoclasts and bone formation by osteoblasts out of the whole life. However, increasedabnormal activity of osteoclasts will lead to bone-resorbing diseases in stomatology such asperi-implantitis and periodontitis. It is a promising method for dental diseases to rebalancebone metabolism by regulating osteoclast function.The forkhead transcription factor family is characterized by a winged-helix DNA bindingmotif and the forkhead domain, with the O class being one of the earliest subfamily to befound. The mammalian forkhead transcription factors of the O class (FOXOs) have fourmembers: FOXO1, FOXO3, FOXO4and FOXO6. FOXOs play important roles in severalphysical and pathological process such as proliferation, apoptosis, autophagy, metabolism,inflammation, differentiation, and stress resistance. FOXO1is the first member of FOXOsubfamily to be found, which is also the most representative for research. Recent researchdemonstrated that FOXO1mediates inflammatory gene expression in both macrophages anddendritic cells, which share a common precursor with osteoclasts. Osteoclasts express all three FOXO isoforms, with FOXO1being, by far, the most abundant member of this familyof proteins. In vivo experiment confirmed that acquire immune response contribute to lyticbone diseases which occurs in the context of increased FOXO1nuclear translocation. In vitro,FOXO1mediates Tumor necrosis factor alpha (TNF-α) induced expression ofpro-osteoclastogenic factors in chondrocytic cells such as Macrophage colony-stimulatingfactor (M-CSF) and Receptor activator of nuclear factor kappa B ligan (RANKL), which islinked to a burst of osteoclast activity and accelerated loss of cartilage in diabetic fractures.Also in old mice, the reduced FOXO1activity in the brain decreases sympathetic tone and, asa result impaires RANKL production in the bone, which further impaires osteoclastdifferentiation and proliferation in old mice bone. So we deduce that FOXO1is related toosteoclastogenesis. However, it is still unclear now whether FOXO1regulateosteoclastogenesis directly and what the mechanism is.In order to investigate function of FOXO1on osteoclastogenesis and bone resorbing, weset up the transgenic mice model with the Cre-LoxP system, in which FOXO1was deletedspecifically in monocyte/macrophage from myeloid progenitors. We crossbred LyzM.Crerecombinase mice with FOXO1L/Lmice, the Cre recombinase on the lysosome gene siterecognized the LoxP site on the FOXO1gene, and FOXO1was then deleted. Cre recombinaseunder the control of a promoter element from Lysosome was used to restrict FOXO1deletionto LyzM+cells. Three different methods (PCR, RT-PCR and immunofluorescence) have beenused for identifying the deletion effeciency of FOXO1in monocyte/macrophage system,which was proved to be up to90%. In the meanwhile, bone marrow-derived macrophages(BMMs) were identified as CD11b positive with immunofluorescence, which proved thatBMMs resulted specifically from BM following M-CSF stimulation. All results proved thatFOXO1was efficiently deleted in BMMs, and the LyzM.Cre+FOXO1L/Ltrangenic micemodel was set up successfully.To establish the bone-resobing model in vivo, experimental mice and control mice wereinjected by soluble RANKL or vehicle PBS into designated area on calvalria, then MicroCTand Tartrate resistant acid phosphatase (TRAP) histostaining were conducted to measure thebone-resorbing ability between two groups. When examined by microCT, more resorption pits appeared on clavarial surface in control group after RANKL injection, the width and area ofsuture also increased significantly (P<0.05). In contrast, fewer resorption pits were observedin experimental group and almost no change was identified in width and area of suturecompared to PBS injection group (P>0.05). TRAP histostaining results suggested that moreTRAP+multinucleated cells appeared in RANKL group compared to PBS group, while inRANKL groups, TRAP+cells number reduced by53%significantly in experimental micecompared to control mice (P<0.05). These results demonstrate that FOXO1deletion inmonocyte/macrophage inhibits osteoclastogenesis in vivo, which leads to reduced boneresorbing ability.TRAP histostaining, F-actin immunostaining, RAW264.7transfection and resorptionactivity assay were utilized to investigate the effect of FOXO1on osteoclastogenesis andresorbing activity of osteoclasts cultured from BMMs and RAW264.7cells in vitro. It isobserved in TRAP histostaining and F-actin immunostaining that deletion of FOXO1resultedin reduction in number, average area and average nuclei of osteoclast (P<0.05). Osteoclastresorption assay demonstrated that fluorescence unit had an up to10-fold increase in RANKLstimulation group compared to that without RANKL treatment in control group(P<0.05),while there is only about1.5-fold increase in experimental group, which suggested that boneresorbing activity reduced significantly with FOXO1deletion. The results prove that FOXO1deletion or silencing plays a negative role in osteoclastogenesis in vitro and inhibits theosteoclast resorbing activity.To determine the expression and nuclear localization of FOXO1in different time pointwith RANKL stimulation, Real-time PCR, immnofluorescence were conducted in BMMs andRAW264.7cells. The mRNA level of FOXO1increased with RANKL treatment and the meanfluorescence intensity (MFI) improved accordingly, which are in a RANKL time-dependentmanner. The transcription activity of FOXO1relys on its nuclear localization. RANKL alsoincreased FOXO1nuclear localization compared to that without RANKL treatment, whichpeaked at day2and declined accordingly at day4. The results demostrate that the expressionand transcription activity of FOXO1is under control of RANKL and enhanced in a timedependent manner in BMMs and RAW264.7cells, the trascription activity peaks at day2, whech is the start of osteoclast precursors fusion.Transwell assay, real-time PCR and immunofluorescence were utilized to obtain insightinto the molecular mechanisms of FOXO1in regulating osteoclastogenesis and its function.The migration of BMMs and RAW264.7cells showed significant increase above the baseline,which is M-CSF dose-dependent, whereas the increase reduced when FOXO1was deleted orsilenced (P<0.05). This suggests that deletion or silencing of FOXO1may inhibit migration ofmacrophages, which plays a negative role in osteocastogenesis. FOXO1nuclear localizationregulated by RANKL was complex and involved MAPK pathway as shown by significantreduction with inhibitors to P38and JNK. The same pathway has been reported in NFATc1transcription activity regulated by RANKL. Following two days’ treatment in BMMs,RANKL stimulated a2.1-fold increase of Nuclear factor activated T cell (NFATc1),32-foldincrease of ATP6v0d2,27-fold increase of Cathepsin K, and929-fold increase of Dendriticcell-specific transmembrane protein (DC-STAMP) at mRNA level (P<0.05), the increase wasdecreased by32%,47%,62%, and55%separately in BMMs from experimental micecompared with control mice (P<0.05). Immunofluorescence results confirmed that RANKLstimulated a19-fold increase of RANK at protein level in BMMs compared with BM, theincrease was decreased by37%in experimental mice compared with conrol mice (P<0.05).2days following RANKL treatment, NFATc1expression also increased significantly (P<0.05).Similar to RANK, the increase of NFATc1was also decreased in experimental mice comparedwith conrol mice (P<0.05). RANKL induced NFATc1nuclear localization apparently,however the increase was decreased by53%in BMMs from experimental mice comparedwith control mice(P<0.05). These experiments demostrate that FOXO1mediateosteoclastogenesis through regulating different moleculars being in charge of migration,differentiation and fusion.Taken together, this research explored role of FOXO1transcription factor inosteoclastogenesis and bone-resorbing function for the first time, and proved that FOXO1mediate osteoclastogenesis through regulating different moleculars. A series of experimentswere conducted in trangenic mice to demonstrate that FOXO1regulate this process directly.Our research provide a new sight to explore the mechanism of osteoclastogenesis and a breakthrough for preveting bone-resorbing dental diseases such as peri-implantitis andperiodontitis...