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PER2-mediated ameloblast differentiation via PPARγ/AKT1/β-catenin axis.

ABSTRACT: Circadian rhythm is involved in the development and diseases of many tissues. However, as an essential environmental regulating factor, its effect on amelogenesis has not been fully elucidated. The present study aims to investigate the correlation between circadian rhythm and ameloblast differentiation and to explore the mechanism by which circadian genes regulate ameloblast differentiation. Circadian disruption models were constructed in mice for in vivo experiments. An ameloblast-lineage cell (ALC) line was used for in vitro studies. As essential molecules of the circadian system, Bmal1 and Per2 exhibited circadian expression in ALCs. Circadian disruption mice showed reduced amelogenin (AMELX) expression and enamel matrix secretion and downregulated expression of BMAL1, PER2, PPARγ, phosphorylated AKT1 and β-catenin, cytokeratin-14 and F-actin in ameloblasts. According to previous findings and our study, BMAL1 positively regulated PER2. Therefore, the present study focused on PER2-mediated ameloblast differentiation and enamel formation. Per2 knockdown decreased the expression of AMELX, PPARγ, phosphorylated AKT1 and β-catenin, promoted nuclear β-catenin accumulation, inhibited mineralization and altered the subcellular localization of E-cadherin in ALCs. Overexpression of PPARγ partially reversed the above results in Per2-knockdown ALCs. Furthermore, in in vivo experiments, the length of incisor eruption was significantly decreased in the circadian disturbance group compared to that in the control group, which was rescued by using a PPARγ agonist in circadian disturbance mice. In conclusion, through regulation of the PPARγ/AKT1/β-catenin signalling axis, PER2 played roles in amelogenin expression, cell junctions and arrangement, enamel matrix secretion and mineralization during ameloblast differentiation, which exert effects on enamel formation.

SUBMITTER: Huang W

PROVIDER: S-EPMC8134554 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Connexin 43 (Cx43) is an integral membrane protein that forms gap junction channels. These channels mediate intercellular transport and intracellular signaling to regulate organogenesis. The human disease oculodentodigital dysplasia (ODDD) is caused by mutations in Cx43 and is characterized by skeletal, ocular, and dental abnormalities including amelogenesis imperfecta. To clarify the role of Cx43 in amelogenesis, we examined the expression and function of Cx43 in tooth development. Single-cell RNA-seq analysis and immunostaining showed that Cx43 is highly expressed in pre-secretory ameloblasts, differentiated ameloblasts, and odontoblasts. Further, we investigated the pathogenic mechanisms of ODDD by analyzing Cx43-null mice. These mice developed abnormal teeth with multiple dental epithelium layers. The expression of enamel matrix proteins such as ameloblastin (Ambn), which is critical for enamel formation, was significantly reduced in Cx43-null mice. TGF-β1 induces Ambn transcription in dental epithelial cells. The induction of Ambn expression by TGF-β1 depends on the density of the cultured cells. Cell culture at low densities reduces cell-cell contact and reduces the effect of TGF-β1 on Ambn induction. When cell density was high, Ambn expression by TGF-β1 was enhanced. This induction was inhibited by the gap junction inhibitors, oleamide, and 18α-grycyrrhizic acid and was also inhibited in cells expressing Cx43 mutations (R76S and R202H). TGF-β1-mediated phosphorylation and nuclear translocation of ERK1/2, but not Smad2/3, were suppressed by gap junction inhibitors. Cx43 gap junction activity is required for TGF-β1-mediated Runx2 phosphorylation through ERK1/2, which forms complexes with Smad2/3. In addition to its gap junction activity, Cx43 may also function as a Ca2+ channel that regulates slow Ca2+ influx and ERK1/2 phosphorylation. TGF-β1 transiently increases intracellular calcium levels, and the increase in intracellular calcium over a short period was not related to the expression level of Cx43. However, long-term intracellular calcium elevation was enhanced in cells overexpressing Cx43. Our results suggest that Cx43 regulates intercellular communication through gap junction activity by modulating TGF-β1-mediated ERK signaling and enamel formation.

Project description:BackgroundStroke is the leading cause of long-term motor disability and cognitive impairment. Recently, neurogenesis has become an attractive strategy for the chronic recovery of stroke. It is important to understand the molecular mechanism that promotes neural stem cell (NSC) neurogenesis for future NSC-based therapies. Our previous study showed that Momordica charantia polysaccharides (MCPs) exerted neuroprotective effects on stroke via their anti-oxidant and anti-inflammation activities. However, it remains unknown whether MCPs promote NSC neurogenesis after cerebral ischemic/reperfusion injury (IRI).MethodsWe investigated MCPs' function in differentiation of neural stem cells (NSCs) in vivo and in vitro experiments. Based on a middle cerebral artery occlusion (MCAO) rat model, the effect of MCPs on neuronal differentiation after MCAO was analyzed. Primary NSCs and neural stem cell line C17.2 were cultured and subjected to glutamate stimulation to establish the cell model of IRI. We evaluated the effect of MCPs on NSC differentiation in IRI cell model by Western blot and immunofluorescence staining. The SIRT1 activity of NSCs post glutamate stimulation was also evaluated by CELL SIRT1 COLORIMETRY ASSAY KIT. In addition, molecular mechanism was clarified by employing the activator and inhibitor of SIRT1.ResultsMCPs had no effects on the differentiation of neural stem cells under physiological conditions while shifted NSC differentiation potential from the gliogenic to neurogenic lineage under pathological conditions. Activation of SIRT1 with MCPs was responsible for the neuronal differentiation of C17.2-NSCs. The neuronal differentiation effect of MCPs was attributed to upregulation SIRT1-mediated deacetylation of β-catenin. MCP-induced deacetylation via SIRT1 promoted nuclear accumulation of β-catenin in NSCs.ConclusionOur findings indicate that the deacetylation of β-catenin by SIRT1 represents a critical mechanism of action of MCPs in promoting NSC neuronal differentiation. It provides an improved understanding of molecular mechanism underlying neuroprotective effects of MCPs in IRI, indicating its potential role on treating ischemic stroke especially chronic recovery.

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PER2-mediated ameloblast differentiation via PPARγ/AKT1/β-catenin axis.

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