Role of CPNE1 in Odontoblastic Differentiation of Rat Stem Cells from Apical Papilla.

CPNE1 is a calcium-dependent, phospholipid-binding protein that is ubiquitously expressed in various tissues and organs. This study investigates the expression and localization of CPNE1 in tooth germ development and the role of CPNE1 in odontoblastic differentiation. In rat tooth germs, CPNE1 is expressed in the odontoblasts and ameloblasts since the late bell stage. The depletion of CPNE1 in the stem cells from apical papilla (SCAPs) clearly inhibits the expression of odontoblastic-related genes and the formation of mineralized nodules during differentiation, while CPNE1 overexpression promotes this process. In addition, CPNE1 overexpression increases AKT phosphorylation during the odontoblastic differentiation of SCAPs. Furthermore, treatment with AKT inhibitor (MK2206) reduces the expression of odontoblastic-related genes in CPNE1 over-expressed SCAPs, and Alizarin Red staining shows reduced mineralization. These results suggest that CPNE1 plays a role in the tooth germ development as well as the odontblastic differentiation of SCAPs in vitro that is related to the AKT signaling pathway.

Cav1.2 regulated odontogenic differentiation of NG2+ pericytes during pulp injury.

NG2+ pericytes, as the possible precursor cells of mesenchymal stem cells (MSCs), have drawn attention due to their ability to differentiate into odontoblasts. Cav1.2 is involved in the differentiation process of stem cells, but its role in the differentiation of NG2+ pericytes is not clear. The aim of the present study was to examine the role of Cav1.2 in the differentiation of NG2+ pericytes into odontoblasts. NG2+ pericytes were obtained from human dental pulp cells by magnetic-activated cell sorting. During the odontogenic differentiation of NG2+ pericytes, the effects of the Cav1.2 inhibitors, nimodipine and Cav1.2 knockdown shRNA, were analyzed by real-time polymerase chain reaction and alizarin red staining. NG2CreERT2/Rosa26-GFP lineage-tracing mice were established to further investigate the roles of NG2+ pericytes and Cav1.2 in incisor self-repair after injury in vivo. At 10 min, 1 day, and 3 days after pulp injuries in transgenic mice, NG2-GFP+ and Cav1.2 immunofluorescence co-staining was performed on the incisors. Nimodipine treatment and Cav1.2 knockdown showed similar inhibition of calcium nodule formation and mRNA levels of osteogenic markers (DSPP, DMP1, and Runx2, p < 0.05). NG2+ pericytes migrated from their inherent perivascular location to the odontoblast layers after pulp injury. Cav1.2 showed a similar response pattern as NG2+ pericytes and gradually returned to normal levels. In addition, many co-stained areas of Cav1.2 and NG2+ pericytes, both near the perivascular and odontoblast layers, were observed. These results indicate that Cav1.2 played a vital role in the odontogenic differentiation of NG2+ pericytes, and that it might be closely linked to the NG2+ pericytes-mediated repair of dental pulp injury in vivo.

NBCe1 Regulates Odontogenic Differentiation of Human Dental Pulp Stem Cells via NF-κB.

Background and Objectives: Dental pulp stem cells (DPSCs) play an important role in the repair of tooth injuries. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a Na＋-coupled HCO3- transporter encoded by the solute carrier 4A4 (SLC4A4) gene and plays a crucial role in maintaining the pH of DPSCs. Our previous research confirmed that NBCe1 is highly expressed in odontoblasts during the development of the tooth germ. Therefore, in this study, we aimed to investigate the effect of NBCe1 on odontogenic differentiation of DPSCs and further clarify the underlying mechanisms. Methods and Results: DPSCs were isolated and identified, and the selective NBCe1 inhibitor S0859 was used to treat DPSCs. We used a cell counting Kit-8 assay to detect cell proliferative ability, and intracellular pH was assessed using confocal microscopy. Odontogenic differentiation of DPSCs was analyzed using real-time PCR and Alizarin Red S staining, and the NF-κB pathway was assessed using western blotting. Our results indicated that 10 µM S0859 was the optimal concentration for DPSC induction. Intracellular pH was decreased upon treatment with S0859. The mRNA expressions of DSPP, DMP1, RUNX2, OCN, and OPN were upregulated in the NBCe1 inhibited group compared to the controls. Moreover, NBCe1 inhibition significantly activated the NF-κB pathway, and a NF-κB inhibitor reduced the effect of NBCe1 on DPSC differentiation. Conclusions: NBCe1 inhibition significantly promotes odontogenic differentiation of DPSCs, and this process may be regulated by activating the NF-κB signaling pathway.

IL-6 Promotes Hepatocellular Carcinoma Invasion by Releasing Exosomal miR-133a-3p.

Interleukin-6 (IL-6), an important inflammatory cytokine, is a key factor regulating cancer metastasis. Cancer cells can modulate their tumorigenic abilities by sorting specific microRNAs (miRNAs) as exosomes into the tumor microenvironment. The relationship between IL-6 and exosomal miRNAs related to hepatocellular carcinoma (HCC) metastasis remains to be elucidated. We examined the metastatic ability of HCC cells after IL-6 treatment and found that miR-133a-3p was sorted into exosomes after IL-6 stimulation and was subsequently released into the tumor microenvironment. In vitro analysis confirmed that exosomal miR-133a-3p acted as a tumor suppressor in HCC. Bioinformatic analysis revealed several signaling pathways and hub genes (CREB1, VCP, CALM1, and YES1) regulated by miR-133a-3p. Survival curves further verified the important roles of hub genes in the prognosis of patients with HCC. It is envisaged that the IL-6/miR-133a-3p axis may be related to the activation of CREB1, VCP, CALM1, and YES1. Our findings provide new insights into the role of exosomal miRNA-mediated tumor progression under inflammatory conditions.

MARCKS on Tumor-Associated Macrophages is Correlated with Immune Infiltrates and Poor Prognosis in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma is the fourth most common cause of cancer-related death. However, the cross-talk between tumor immune microenvironment and hepatocellular carcinoma (HCC) remains unclear. MATERIAL AND METHODS: We analyzed the expression of miR-143-3p in exosomes from different HCC cell lines. Differentially expressed genes (DEGs) in Tumor-associated macrophages (TAMs) co-cultured with HCC cell lines were overlapped with miR-143-3p target genes. We used the Oncomine, Kaplan-Meier plotter, and The Cancer Genome Atlas (TCGA) databases to assess Myristoylated alanine-rich C-kinase substrate (MARCKS) expression in various types of cancers. The relationship between patient clinicopathological characteristics and MARCKS expression level was identified using the Kaplan-Meier plotter database. Last, we analyzed how MARCKS expression correlated with immune infiltration makers using the TCGA database, Tumor IMmune Estimation Resource (TIMER), and Gene Expression Profiling Interactive Analysis (GEPIA). RESULTS: Exosomal miR-143-3p was elevated after IL-6 treatment in the HCC cell line. MARCKS, a target gene of miR-143-3p, was up-regulated in Tumor-associated macrophages co-cultured with high-metastatic-potential HCC cell line. MARCKS expression was identified as significantly correlated with outcome in multiple types of cancer, especially in HCC. High MARCKS expression level was associated with poorer overall survival (OS), Progress-free survival (PFS), and also with patient gender, race, hepatitis virus background, stage, grade, AJCC_T, and vascular invasion. MARCKS was positively associated with levels of T follicular helper cells (TFH) (R = .48, p < .001), T helper type 2 (Th2) cells (R = .47, p < .001), macrophages (R = .41, p ≤ .001), T helper cells (R = .40, p < .001), T helper type 1 (Th1) cells (R = .38, p < .001), T cells (R = .34, p < .001), NK CD56bright cells (R = .34, p < .001) and immature DC (iDC) (R = .33, p < .001), and negatively associated with levels of T helper 17 (Th17) cells. Also, MARCKS may influence the M2 polarization and immune escape. CONCLUSION: The present study suggests that MARCKS on TAMs is associated with poor prognosis and immune cell infiltration in HCC.

Exosomes derived from lipopolysaccharide-preconditioned human dental pulp stem cells regulate Schwann cell migration and differentiation.

Purpose: Schwann cells (SCs) are the main source of odontoblasts. They can migrate to the sites of injury and differentiate into odontoblasts during tooth development and regeneration. However, the molecular mechanisms by which SCs repair dental damage remain to be fully elucidated. In addition, exosomes play a crucial role in regulating cell-cell interaction. Hence, we aim to explore the biological function of exosomes secreted by human dental pulp stem cells (hDPSCs) and their effect on SCs.Materials and Methods: Exosomes were extracted from the supernatant of hDPSCs (exo) and LPS- preconditioned hDPSCs (LPS-exo), respectively. Following the evaluation of specific surface proteins and exosomes size and morphology, SCs were treated with exo and LPS-exo, and we examined SCs proliferation, migration, and odontogenic differentiation in vitro.Results: Exosomes had the capacity to regulate SCs proliferation and migration. Furthermore, exosomes from both groups stimulated SCs to produce dentin sialoprotein and undergo mineralization; however, LPS-exo had a better ability to modulate SCs migration and odontogenic differentiation compared with exo.Conclusions: Exosomes from hDPSCs, especially from LPS- preconditioned hDPSCs, can promote the proliferation, migration and odontogenic differentiation of SCs. LPS might change the hDPSCs' intercellular signals, which might mediate the odontogenic differentiation of SCs, transmitting in the manner of "exosomes".

Transcriptional Analysis Reveals Key Genes in the Pathogenesis of Nifedipine-Induced Gingival Overgrowth.

BACKGROUND: Nifedipine-induced gingival overgrowth (NGO) is a multifactorial pathogenesis with increased extracellular matrix including collagen and glycans, inflammatory cytokines, and phenotype changes of fibroblasts. However, the molecular etiology of NGO is not well understood. The objective of this study is to investigate the key genes in the pathogenesis of NGO. METHODS: In this study, we examined the proliferation and migration abilities of fibroblasts derived from patients with chronic periodontitis, nifedipine nonresponder gingival overgrowth, gingival overgrowth caused by nifedipine, and healthy normal gingiva. We conducted RNA-Seq on these four groups of fibroblasts and analysed the differentially expressed genes (DEGs). RESULTS: Fibroblasts derived from NGO patients had higher proliferation and migration abilities than those of the other groups. Protein-protein interaction network analysis indicated that TGFB2, ITGA8, ITGA11, FGF5, PLA2G4D, PLA2G2F, PTGS1, CSF1, LPAR1, CCL3, and NKX3-1 are involved in the development of NGO. These factors are related to the arachidonic acid metabolism and PI3K/AKT signaling pathways. CONCLUSION: Transcriptional gene expression analysis identified a number of DEGs that might be functionally related to gingival overgrowth induced by nifedipine. Our study provides important information on the molecular mechanism underlying nifedipine-induced gingival overgrowth.

Distal C-terminus of Cav 1.2 is indispensable for the chondrogenic differentiation of rat dental pulp stem cells.

The α1 subunit (Cav1.2) of the L-type calcium channel (LTCC), which is presently existing in both excitatory cells and non-excitatory cells, is involved in the differentiation and proliferation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs), MSCs derived from dental pulp, exhibit multipotent characteristics similar to those of MSCs. The aim of the present study was to examine the contribution of Cav1.2 and its distal C-terminus (DCT) to the commitment of rat DPSCs (rDPSCs) toward chondrocytes and adipocytes in vitro. The expression of Cav1.2 was obviously elevated in chondrogenic differentiation but did not differ significantly in adipogenic differentiation. The chondrogenic differentiation but not adipogenic of rDPSCs was inhibited by either blocking LTCC using nimodipine or knockdown of Cav1.2 via short hairpin RNA (shRNA). Overexpression of DCT rescued the inhibition by Cav1.2-shRNA during chondrogenic differentiation, indicating that DCT is essential for the chondrogenic differentiation of rDPSCs. However, the protein level of DCT decreased after chondrogenic differentiation in wild-type cells, and overexpression of DCT in rDPSCs inhibited the phenotype. These data suggest that DCT is indispensable for chondrogenic differentiation of rDPSCs but that superfluous DCT inhibits this process. Through the analysis of differentially expressed genes using RNA-seq data, we speculated that the regulation of DCT might be mediated by the mitogen-activated protein kinase/extracellular-regulated kinase and c-Jun N-terminal kinase signaling pathways, or Chondromodulin-1.

Lipopolysaccharide upregulates the proliferation, migration, and odontoblastic differentiation of NG2+ cells from human dental pulp in vitro.

NG2+ cells have been proven to differentiate into odontoblasts in vivo, and their contribution to odontoblasts is significantly increased, especially after tooth injury. However, their characteristics in vitro, especially under an inflammatory environment, are still not fully understood. Therefore, this study aimed to explore their proliferation, migration, and odontoblastic differentiation ability after treatment with lipopolysaccharide (LPS) in vitro. In our study, NG2 + cells were isolated from the human dental pulp by magnetic-activated cell sorting, and these isolated cells were proven to be NG2 + by immunostaining. When compared with human dental pulp cells (hDPCs), the NG2 + cells showed no significant differences in cell migration with or without LPS incubation, but their proliferative ability was weaker. When treated with LPS, NG2 + cells expressed elevated levels of pro-inflammatory cytokines including interleukin-1ß (IL-1ß), IL-6, IL-8, and tumor necrosis factor-α, and among these, the expression of IL-1ß and IL-6 were higher than that of hDPCs. Their multipotent differentiation potential was confirmed by the induction of odontoblastic and adipogenic differentiation, and LPS increased their odontoblastic differentiation capacity. In the odontoblastic differentiation process, Wnt5a, BMP2, and BMP7 mRNA were increased, while the canonical Wnt-related genes were decreased. In conclusion, the LPS stimulation promotes the migration, proliferative, and odontoblastic differentiation ability of NG2 + cells from the human dental pulp in vitro, and bone morphogenetic protein and the noncanonical Wnt pathway may be involved in their odontoblastic differentiation. These results indicated their special roles in tooth injury repair and potential application in pulp regeneration.

Roles of L-type calcium channels (CaV1.2) and the distal C-terminus (DCT) in differentiation and mineralization of rat dental apical papilla stem cells (rSCAPs).

OBJECTIVE: Voltage-gated inward Ca2+ currents (ICa) are triggered by cell depolarization and commonly produce transient increases in the cytoplasmic free Ca2+ concentration. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt or DCT). Stem cells from the apical papilla (SCAPs) has a capacity for differentiation into the odontoblastic-like cells in vitro and dentin forming in vivo, which makes SCAPs advantages in tissue engineering and regenerative endodontic. The aim of this study was to investigate the effect of CaV1.2 and its distal C-terminal fragment in the odontoblastic differentiation of rat SCAPs (stem cells from the apical papilla). DESIGN: In this study, we generated stable CaV1.2 knockdown and DCT over-expressed rSCAPs using short hairpin RNA and DCT gene containing Lentivirus vectors, respectively. The transfected apical papilla cells were induced to differentiate into the odontoblast-like cells, and the expression of markers for odontoblastic differentiation were analyzed by alizarin red staining, Real-time Polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: The knockdown of CaV1.2 and excess expression of DCT both suppressed the expression of DSPP, ALP in mRNA level and the formation of calcium nodules. CONCLUSIONS: Our results suggest that CaV1.2 and DCT play important roles in the differentiation of rSCAPs, DCT might act as a transcription factor and regulate the differentiation of rSCAPs.

Cav1.2 of L-type Calcium Channel Is a Key Factor for the Differentiation of Dental Pulp Stem Cells.

INTRODUCTION: L-type calcium channel (LTCC) is a unique and important factor in several cell lineages, whereas its role in the differentiation of dental pulp stem cells (DPSCs) is not well-known. In this study, we examined the function of LTCC α1C subunit (Cav1.2) and its distal C-terminus (DCT) during the in vitro differentiation of rat DPSCs (rDPSCs). METHODS: After fluorescence-activated cell sorting, rDPSCs were differentiated toward dentin sialophosphoprotein-positive odontoblasts and neural cells expressing specific neuronal markers. The inhibition of rDPSC differentiation via LTCC blocker nimodipine and Cav1.2 knockdown through short hairpin RNA was evaluated by using quantitative real-time polymerase chain reaction, Western blot, and immunofluorescence staining. RESULTS: Nimodipine treatment and Cav1.2 knockdown generated similar results. The number of positive calcium nodules and the protein and mRNA levels of dentin sialophosphoprotein were significantly reduced during odontogenic differentiation. The levels of microtubule-associated protein-2 and ß-III-tubulin were reduced in neural differentiation. The expression of DCT decreased after odontogenic differentiation but significantly increased after neural differentiation (P < .05, n = 9). CONCLUSIONS: Our data showed that LTCC blocker nimodipine inhibits the odontogenic and neural differentiation of rDPSCs, and Cav1.2 is responsible for the activity of LTCC. The expression of DCT of Cav1.2 significantly changes during both odontogenic and neural differentiation. Thus, Cav1.2 of LTCC plays an essential role in differentiation of DPSCs, which might be mediated through the regulation of DCT levels in DPSCs.

Distal C terminus of CaV1.2 channels plays a crucial role in the neural differentiation of dental pulp stem cells.

L-type voltage-dependent CaV1.2 channels play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. C-terminal cleavage of CaV1.2 channels was reported in several types of excitable cells, but its expression and possible roles in non-excitable cells is still not clear. The aim of this study was to determine whether distal C-terminal fragment of CaV1.2 channels is present in rat dental pulp stem cells and its possible role in the neural differentiation of rat dental pulp stem cells. We generated stable CaV1.2 knockdown cells via short hairpin RNA (shRNA). Rat dental pulp stem cells with deleted distal C-terminal of CaV1.2 channels lost the potential of differentiation to neural cells. Re-expression of distal C-terminal of CaV1.2 rescued the effect of knocking down the endogenous CaV1.2 on the neural differentiation of rat dental pulp stem cells, indicating that the distal C-terminal of CaV1.2 is required for neural differentiation of rat dental pulp stem cells. These results provide new insights into the role of voltage-gated Ca(2+) channels in stem cells during differentiation.