The Roles And Underlying Mechanisms Of Leukemia Inhibitory Factor On Enamel Development

PartⅠ:Spatiotemporal Expression of LIF During Tooth DevelopmentAims:To investigate the spatiotemporal distribution of LIF in cells during mouse tooth morphogenesis.Material&Methods:Embryos were collected on days 13.5,15.5,16.5,and 18.5 from pregnant mice by euthanization.3 days postnatal newborn mice were euthanized and the mandibular were dissected.Following pretreatment of decalcification and sectioned,immunohistochemistry staining was performed to detect the expression of LIF in tooth germ.Results:According to immunohistochemistry staining,no expression of LIF was observed in the bud-stage,early-cap-stage,or late-cap-stage tooth germs.In the bell-stage tooth germs,LIF expression was observed in cells of the inner enamel epithelium and dental follicle at embryonic-day 18.5.At the 3 days postnatal,LIF was expressed in odontoblasts and stellate reticulum cells and highly expressed in ameloblasts.Conclusion:LIF was expressed in tooth germs at bell-stage and postnatal 3 days,indicating that LIF might be involved in enamel development and play a physiological role.PartⅡ:The Phenotypes of Enamel in Lif-knockout MiceAims:To investigate the effects of LIF deficiency on enamel development.Material&Methods:By generating Lif heterozygote mice(Lif^+/-mice),Lif-knockout mice(Lif^-/-)were obtained from heterozygous-heterozygous mating.Wild-type littermates were used as controls in all experiments.The genotype of the mouse was identified by PCR and immunohistochemistry.SEM was performed for surface morphology and microstructure observation.The mandibles were scanned by aμCT instrument and mineral density was analyzed by CT analysis software.Microhardness measurement was performed at different layers of incisors.The calcium concentration of the leaching solution of incisors was detected.EDS was performed for surface elements analysis of incisors.Prussian blue staining,an assessment of iron deposition,was performed to further confirm the iron content in ameloblasts.Results:Lif^-/-mice were obtained from heterozygous-heterozygous mating.It was found that incisors of Lif-knockout mice lacked pigmentation,exhibiting a much whiter color than those of wild-type and heterozygous littermates.LIF was mainly localized at the basal side of the cytoplasm of ameloblasts of control mice.No expression of LIF was observed in maturation-stage ameloblasts of Lif^-/-mice.Although there were no structural abnormalities or defective mineralization according to SEM and CT analysis,three-dimensional images showed that the length of incisors was shorter in Lif^-/-mice.Microhardness and acid resistance assays showed that the Vickers hardness and acid resistance of the enamel surface of Lif^-/-mice were decreased compared to those of wild-type mice.The iron content of the incisors was significantly reduced,as confirmed by EDS and Prussian blue staining.Conclusion:LIF deficiency led to decreased iron deposition on the enamel surface,resulting in a whitish appearance with decreased hardness and acid resistance.PartⅢ:The Molecular Mechanism by which LIF Functions in Enamel DevelopmentAims:The functions of LIF during tooth development and the underlying mechanisms involved in this process were explored.Material&Methods:Serum collected from the blood by centrifuging was used for the analysis of serum iron parameters.Hematoxylin-eosin staining of the slice was performed for cellular morphology observation.q RT-PCR and western blot were performed to detect the expression of relevant genes of secretory-stage ameloblasts,maturation-stage ameloblasts.Lif-knockdown efficiency and iron transportation-related genes were determined by q RT-PCR and western blot assays.ALC was treated with different concentrations of recombined LIF,or treated with 20 ng/m L Lif with or without Stattic.The expression of transportation-related genes and activation of relevant signaling pathways was confirmed by western blot.To investigate the functions of LIF on ALC during osteogenic induction,ALP staining,alizarin staining,q RT-PCR,and western blot were performed.Results:The serum iron concentration of Lif^-/-mice was comparable to that of wild-type littermates.Histological staining showed that the cell length of maturation-stage ameloblasts was shorter in Lif^-/-mice.Levels of Amelx,Ambn,Enam,and Klk4 and expression of AMELX in secretory-stage ameloblasts were comparable between the two genotypes.q RT-PCR revealed that transcriptional levels of iron transportation-related genes,Tfrc,Slc40a1,and Ftl were significantly reduced in Lif^-/-mice.Moreover,expression of TFRC and SLC40A1 were decreased in maturation-stage ameloblasts of Lif^-/-mice compared to those of control mice.In vitro,by generating ALC-Lif-Sh in which the expression of LIF was knockdown by over 65%compared to the Control-sh group,decreased iron transport-related gene expression was observed,in agreement with the results of immunohistochemistry and q RT-PCR in Lif^-/-mice.Besides,western blot results showed that expression of TFRC and SLC40A1 was upregulated in ALC treated with 10ng/m L and 20 ng/m L LIF,accompanied by the STAT3 signaling pathway activation.Stattic administration effectively inhibited STAT3 phosphorylation stimulated by LIF.In addition,upregulation of TFRC and SLC40A1 by LIF was mitigated by Stattic stimulation.Although the ALP staining was not significantly different,Alizarin staining showed that LIF stimulated the osteogenic ability of ALC with upregulation of OPN and OCN expression confirmed by q RT-PCR and western blot.Conclusion:LIF regulates the expression of TFRC and SLC40A1 in ALC in a STAT3signaling pathway-dependent manner,which modulates the process of iron transportation.Meanwhile,LIF is capable of upregulating the expression of OPN and OCN in ALC,with the formation of mineralization nodules promoted.