GSK3β rephosphorylation rescues ALPL deficiency-induced impairment of odontoblastic differentiation of DPSCs.
Ontology highlight
ABSTRACT: Background
Premature exfoliation of the deciduous teeth is a common manifestation in childhood patients with hypophosphatasia (HPP), which is an autosomal inherited disease caused by ALPL mutations. Dysplasia of the cementum, dentin, and alveolar bone has been proposed to be the main reasons for the exfoliation of teeth, while the extraordinarily complex intracellular mechanisms remain elusive. Dental pulp stem cells (DPSCs) have been demonstrated to successfully regenerate functional pulp-dentin-like tissue. Dental pulp cells derived from HPP patients impaired mineralization; however, insight into the deeper mechanism is still unclear.Methods
The effects of ALPL on odontoblastic differentiation of DPSCs from HPP patient were assessed by Alizarin Red staining, immunofluorescent staining, Western blot and RT-PCR, and micro-CT assays.Result
Here, we found DPSCs from HPP patient exhibited low ALP activity and impaired odontoblastic differentiation. Meanwhile, we found that loss of function of ALPL reduced phosphorylation of GSK3β in DPSCs. While GSK3β rephosphorylation improved odontoblastic differentiation of HPP DPSCs with LiCl treatment. Finally, we demonstrated systemic LiCl injection ameliorated tooth-associated defects in ALPL+/- mice by enhanced phosphorylation of GSK3β in the teeth.Conclusions
Our study indicates that ALPL regulates odontoblastic differentiation of DPSCs and provides useful information for understanding how ALPL deficiency led to tooth dysplasia and, ultimately, may inform efforts at improvement tooth defects in HPP patients.
SUBMITTER: Zhang L
PROVIDER: S-EPMC8022410 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The presence of stem cells within the dental-pulp tissue as well as their differentiation into a new generation of functional odontoblast-like cells constitutes an important step of the dentin-pulp regeneration. Recent investigations demonstrated that the complement system activation participates in 2 critical steps of dentin-pulp regeneration: pulp progenitor's recruitment and pulp nerve sprouting. Surprisingly, its implication in odontoblastic differentiation has not been addressed yet. Since the complement receptor C5a receptor-like 2 (C5L2) is expressed by different stem cells, the aim of this study is to investigate if the dental pulp stem cells express C5L2 and if this receptor participates in odontoblastic differentiation. Immunohistochemistry performed on human third molar pulp sections showed a perivascular co-localization of the mesenchymal stem cell markers STRO1 and C5L2. In vitro immunofluorescent staining confirmed that hDPSCs express C5L2. Furthermore, we determined by real-time polymerase chain reaction that the expression of C5L2 is highly modulated in human dental pulp stem cells (hDPSCs) undergoing odontoblastic differentiation. Moreover, we showed that this odontogenesis-regulated expression of C5L2 is specifically potentiated by the proinflammatory cytokine TNFα. Using a C5L2-siRNA silencing strategy, we provide direct evidence that C5L2 constitutes a negative regulator of the dentinogenic marker DMP1 (dentin matrix protein 1) expression by hDPSCs. Our findings suggest a direct correlation between the odontoblastic differentiation and the level of C5L2 expression in hDPSCs and identify C5L2 as a negative regulator of DMP1 expression by hDPSCs during the odontoblastic differentiation and inflammation processes. This work is the first to demonstrate the involvement of C5L2 in the biological function of stem cells, provides an important knowledge in understanding odontoblastic differentiation of dental pulp stem cells, and may be useful in future dentin-pulp engineering strategies.
| S-EPMC6481006 | biostudies-literature
Project description:ATF6 is an endoplasmic reticulum (ER) membrane-bound transcription factor that regulates various cellular functions. The purpose of this study was to investigate the role of ATF6 in odontoblast differentiation. Rat tooth germs were isolated, changes in gene expression were evaluated over time, and localization of ATF6 was determined by immunohistochemistry. Human dental pulp cells (HDPCs) were cultured with 50 µg/mL ascorbic acid and 5 mmol/L ?-glycerophosphate or 100 ng/mL bone morphogenetic protein 2 to induce differentiation. Translocation of ATF6 was observed by immunofluorescence and confocal microscopy. Overexpression of ATF6 was performed with an adenoviral vector. Matrix mineralization was evaluated by alizarin red staining. Immunoreactivity to anti-ATF6 was observed in the odontoblastic layer of the molar tooth germ, and expressions of ATF6, dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) increased gradually during tooth germ development. When HDPCs were cultured in differentiation media, ATF6, DSPP, and DMP1 expression increased with the expression of unfolded protein response (UPR) markers, BiP and CHOP. Immunofluorescence results showed that ATF6 protein moved from cytoplasm to nucleus when cells were exposed to differentiation media. Notably, overexpression of ATF6 increased DSPP and DMP1 expression, alkaline phosphatase (ALP) activity, and matrix mineralization in HDPC cultures. Inhibition of ATF6 decreased ALP activity and mineralization. These results suggest that ER membrane-bound transcriptional factor ATF6 may be involved in odontoblastic differentiation.
| S-EPMC6728569 | biostudies-literature
Project description:Feeding rodents a high-fat diet (HFD) disrupts normal behavioral rhythms, particularly meal timing. Within the brain, mistimed feeding shifts molecular rhythms in the hippocampus and impairs memory. We hypothesize that altered meal timing induced by an HFD leads to cognitive impairment and that restricting HFD access to the "active period" (i.e., night) rescues the normal hippocampal function. In male mice, ad-lib access to an HFD for 20 weeks increased body weight and fat mass, increased daytime meal consumption, reduced hippocampal long-term potentiation (LTP), and eliminated day/night differences in spatial working memory. Importantly, two weeks of time-restricted feeding (TRF) at the end of the chronic HFD protocol rescued spatial working memory and restored LTP magnitude, even though there was no change in body composition and total daily caloric intake. These findings suggest that short-term TRF is an effective mechanism for rescuing HFD-induced impaired cognition and hippocampal function.
| S-EPMC8188491 | biostudies-literature
Project description:Dentin phosphophoryn synthesized and processed predominantly by the odontoblasts, functions as both structural and signaling protein. Mechanistic studies revealed that DPP stimulation of DPSCs positively impacted the differentiation of DPSCs into functional odontoblasts. Results show that NF-κB signaling and transcriptional activation of genes involved in odontoblast differentiation were influenced by DPP signaling. Specifically, RelA/p65 subunit of NF-κB was identified as being responsible for the initiation of the differentiation cascade. Confocal imaging demonstrated the nuclear translocation of p65 with DPP stimulation. Moreover, direct binding of nuclear NF-κB p65 subunit to the promoter elements of Runx2, Osx, OCN, MMP1, MMP3, BMP4 and PTX3 were identified by ChIP analysis. Pharmacological inhibition of the NF-κB pathway using TPCA-1, a selective inhibitor of IKK-2 and JSH-23, an inhibitor that prevents nuclear translocation and DNA binding of p65 showed impairment in the differentiation process. Functional studies using Alizarin-Red staining showed robust mineral deposits with DPP stimulation and sparse deposition with defective odontoblast differentiation in the presence of inhibitors. In vivo expression of NF-κB targets such as OSX, OCN, PTX3 and p65 in odontoblasts and dental pulp cells from DSPP null mouse was lower when compared with the wild-type. Overall, the results suggest an important role for DPP-mediated NF-κB activation in the transcriptional regulation of early odontogenic markers that promote differentiation of DPSCs.
| S-EPMC8586344 | biostudies-literature
Project description:BACKGROUND AND PURPOSE:Alzheimer's disease (AD) is a common neurodegenerative disease characterized by a neuroinflammatory state, and to date, there is no cure and its treatment represents a large unmet clinical need. The involvement of Th17 cells in the pathogenesis of AD-related neuroinflammation has been reported in several studies. However, the role of the cytokine, IL-17 has not been well addressed. Herein, we investigate the effects of IL-17 neutralizing antibody (IL-17Ab) injected by i.c.v. or intranasal (IN) routes on amyloid-? (A?)-induced neuroinflammation and memory impairment in mice. EXPERIMENTAL APPROACH:A?1-42 was injected into cerebral ventricles of adult CD1 mice. These mice received IL-17Ab via i.c.v. either at 1 h prior to A?1-42 injection or IN 5 and 12 days after A?1-42 injection. After 7 and 14 days of A?1-42 administration, we evaluated olfactory, spatial and working memory and performed biochemical analyses on whole brain and specific brain areas. KEY RESULTS:Pretreatment with IL-17Ab, given, i.c.v., markedly reduced A?1-42 -induced neurodegeneration, improved memory function, and prevented the increase of pro-inflammatory mediators in a dose-dependent manner at 7 and 14 days. Similarly, the double IN administration of IL-17Ab after A?1-42 injection reduced neurodegeneration, memory decline, and the levels of proinflammatory mediators and cytokines. CONCLUSION AND IMPLICATIONS:These findings suggest that the IL-17Ab reduced neuroinflammation and behavioural symptoms induced by A?. The efficacy of IL-17Ab IN administration in reducing A?1-42 neurodegeneration points to a possible future therapeutic approach in patients with AD. LINKED ARTICLES:This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.
| S-EPMC6715610 | biostudies-literature
Project description:Efferocytosis, a process of clearance of apoptotic cells by phagocytes, is essential for successful resolution of inflammation and maintenance of tissue homeostasis. Diabetes compromises the function of macrophages leading to adverse inflammatory response during wound healing, myocardial injury, atherosclerosis and autoimmune disorders. However, the effect of diabetes on macrophage-mediated efferocytosis of apoptotic cardiomyocytes (ACM) and the molecular mechanisms involved are not understood so far. In the present study we found that invitro efferocytosis of ACM was impaired in macrophages from db/db (diabetic) mice. Macrophages exposed to high glucose (HG) decreases microRNA-126 (miR-126) expression with a corresponding increase in ADAM9 expression. Dual-luciferase reporter assay confirms that ADAM9 3'UTR contains miR-126 target site. ADAM9 inhibition reduces HG-induced proteolytic cleavage of Mer tyrosine receptor kinase (MerTK, a proto-oncogene that plays a critical role in phagocytosis), resulting in shedding of soluble-Mer (sMER) and loss of MERTK function. Over-expression of miR-126 attenuates HG-induced impairment of efferocytosis. Furthermore, human diabetic hearts show lower miR-126 expression with a corresponding increase in ADAM9 expression vs. normal counterparts. These data suggests that diabetes impairs efferocytosis of ACM and that strategies to enhance efferocytosis might attenuate diabetes-induced impairment in inflammation resolution and cardiac repair after injury.
| S-EPMC5101812 | biostudies-literature
Project description:Dental pulp tissue contains many undifferentiated mesenchymal cells, which retain the ability to differentiate into mature cells. Induced pluripotent stem cells have been developed from various cell sources, including dental pulp-derived stem cells, and evaluated for potential application to regenerative therapy. Dental pulp tissues overexpress CD44, a cell-adhesion factor involved in the induction of mineralization. In this study, we investigated the effects of hyaluronan-a known CD44 ligand-on dental pulp stem cells (DPSCs).DPSC CD44 expression was analyzed using immunofluorescence staining, flow cytometry, and western blotting. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Effects of hyaluronan on the cell cycle were analyzed by flow cytometry. Alkaline phosphatase activity was employed as marker of mineralization and measured by fluorometric quantification and western blotting. Bone morphogenetic protein (BMP)-2, BMP-4, dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein 1 (DMP-1) levels were measured using real-time polymerase chain reaction. Odontoblastic differentiation and the close cell signaling examination of DPSC differentiation were determined using western blotting.Hyaluronan induced expression of the odontoblastic differentiation markers DMP-1 and DSPP. Moreover, the odontoblastic differentiation induced by hyaluronan was mediated by CD44-but not by Akt, Smad1 or MAPK signaling.Our results indicate that hyaluronan induces odontoblastic differentiation of DPSCs via CD44. This suggests that hyaluronan plays a crucial role in the induction of odontoblastic differentiation from DPSCs. Our findings may aid the development of new, inexpensive, and effective conservative treatments for dental pulp repair.
| S-EPMC5029108 | biostudies-literature
Project description:Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.
| S-EPMC8267829 | biostudies-literature
Project description:Odontoblasts are post-mitotic cells responsible for maintenance of the dentin, and are therefore important for dental health. In some cases, irreversible pulpitis leads to necrosis and consequently death of odontoblasts. Regenerative endodontics (RE) uses the concept of tissue engineering to restore the root canals to a healthy state, allowing for continued development of the root and surrounding tissue. Human dental pulp stem cells (hDPSCs) have been successfully used in RE to restore odontoblast function. Surface microgeometry is one of the most important factors involved in the induction of differentiation of hDPSCs into odontoblast-like cells. Although different authors have demonstrated the importance of a dentin-like surface with accessible dentin tubules to induce differentiation of hDPSCs, the ultrastructural characteristics of the cells and the secreted extracellular matrix have not been studied in depth. Here, we used an acellular dentin scaffold containing dentin tubules in different spatial geometries, which regulated their accessibility to cells. hDPSCs were cultured on the scaffolds for up to 6 weeks. Systematic characterization of differentiated cells was performed using both optical (hematoxylin and eosin, Masson trichrome, and immunohistochemical determination of dentin sialoprotein [DSSP]) and transmission electron microscopy. The results presented here indicated that cells grown on the dentin surface containing accessible dentin tubules developed a characteristic odontoblastic phenotype, with cellular processes similar to native odontoblasts. The cell organization and characteristics of secreted extracellular matrix were also similar to those of native dentin tissue. Cells grown on non-accessible dentin tubule surfaces secreted a more abundant and dense extracellular matrix, and developed a different phenotype consisting of secretory flat cells organized in layers. Cells grown far from the scaffold, i.e., directly on the culture well surface, developed a secretory phenotype probably influenced by biochemical factors released by the dentin scaffold or differentiated cells. The results presented here support the use of hDPSCs to regenerate dentin and show the utility of scaffold microgeometry for determining the differentiation and secretory phenotype of cultured cells.
| S-EPMC6508697 | biostudies-literature
Project description:Brain serotonin (5-HT) system dysfunction is implicated in depressive disorders and acute depletion of 5-HT precursor tryptophan has frequently been used to model the influence of 5-HT deficiency on emotion regulation. Tamoxifen (TAM)-induced Cre/loxP-mediated inactivation of the tryptophan hydroxylase-2 gene (Tph2) was used to investigate the effects of provoked 5-HT deficiency in adult mice (Tph2 icKO) previously subjected to maternal separation (MS). The efficiency of Tph2 inactivation was validated by immunohistochemistry and HPLC. The impact of Tph2 icKO in interaction with MS stress (Tph2 icKO × MS) on physiological parameters, emotional behavior and expression of 5-HT system-related marker genes were assessed. Tph2 icKO mice displayed a significant reduction in 5-HT immunoreactive cells and 5-HT concentrations in the rostral raphe region within four weeks following TAM treatment. Tph2 icKO and MS differentially affected food and water intake, locomotor activity as well as panic-like escape behavior. Tph2 icKO prevented the adverse effects of MS stress and altered the expression of the genes previously linked to stress and emotionality. In conclusion, an experimental model was established to study the behavioral and neurobiological consequences of 5-HT deficiency in adulthood in interaction with early-life adversity potentially affecting brain development and the pathogenesis of depressive disorders.
| S-EPMC7940624 | biostudies-literature