RICK regulates the odontogenic differentiation of dental pulp stem cells through activation of TNF-α via the ERK and not through NF-κB signaling pathway

Dental pulp stem cells (DPSCs) are capable of both self-renewal and multilineage differentiation, which play a positive role in dentinogenesis. Studies have shown that tumor necrosis factor-α (TNF-α) is involved in the differentiation of DPSCs under pro-inflammatory stimuli, but the mechanism of action of TNF-α is unknown. Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) is a biomarker of an early inflammatory response that plays a key role in modulating cell differentiation, but the role of RICK in DPSCs is still unclear. In this study, we identified that RICK regulates TNF-α-mediated odontogenic differentiation of DPSCs via the ERK signaling pathway. The expression of the biomarkers of odontogenic differentiation dental matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), biomarkers of odontogenic differentiation, increased in low concentration (1–10 ng/ml) of TNF-α and decreased in high concentration (50–100 ng/ml). Odontogenic differentiation increased over time in the odontogenic differentiation medium. In the presence of 10 ng/L TNF-α, the expression of RICK increased gradually over time, along with odontogenic differentiation. Genetic silencing of RICK expression reduced the expression of odontogenic markers DMP-1 and DSPP. The ERK, but not the NF-κB signaling pathway, was activated during the odontogenic differentiation of DPSCs. ERK signaling modulators decreased when RICK expression was inhibited. PD98059, an ERK inhibitor, blocked the odontogenic differentiation of DPSCs induced by TNF-α. These results provide a further theoretical and experimental basis for the potential use of RICK in targeted therapy for dentin regeneration.     

Estrogen deficiency inhibits the odonto/osteogenic differentiation of dental pulp stem cells via activation of the NF-κB pathway

Various factors can affect the functions of dental pulp stem cells (DPSCs). However, little knowledge is available about the effects of estrogen deficiency on the differentiation of DPSCs. In this study, an estrogen-deficient rat model was constructed and multi-colony-derived DPSCs were obtained from the incisors of ovariectomized (OVX) or sham-operated rats. Odonto/osteogenic differentiation and the possible involvement of the nuclear factor kappa B (NF-κB) pathway in the OVX-DPSCs/Sham-DPSCs of these rats were then investigated. OVX-DPSCs presented decreased odonto/osteogenic capacity and an activated NF-κB pathway, as compared with Sham-DPSCs. When the cellular NF-κB pathway was specifically inhibited by BMS345541, the odonto/osteogenic potential in OVX-DPSCs was significantly upregulated. Thus, estrogen deficiency down-regulated the odonto/osteogenic differentiation of DPSCs by activating NF-κB signaling and inhibition of the NF-κB pathway effectively rescued the decreased differentiation potential of DPSCs.     

Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCγ signaling pathway

Basic fibroblast growth factor (basic FGF) has pivotal roles in the function of various cell types. Here, we report the effects of basic FGF in the regulation of dental pulp stem cell (DPSC) behaviors including maintaining stemness and directing differentiation. Cells isolated from human dental pulp tissues exhibited stem cell properties including the expression of mRNA markers for embryonic and mesenchymal stem cells, the expression of Stro-1, and the multipotential differentiation. Basic FGF stimulated colony-forming units of DPSCs and up-regulated the expression of the embryonic stem cell markers; Oct4, Rex-1, and Nanog. Moreover, osteogenic medium containing basic FGF inhibited alkaline phosphatase enzymatic activity and mineralization of DPSCs. On the contrary, basic FGF appeared to be an influential growth factor in the neurogenic differentiation of DPSCs. In the presence of basic FGF, increased DPSCs neurosphere size and the up-regulation of neurogenic markers were noted. Inhibitors of FGFR or PLCγ were able to ablate the basic FGF-induced neuronal differentiation of DPSCs. Taken together, these results suggest basic FGF may be involved in the mechanisms controlling DPSCs cell fate decisions.     

Mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation

Objective: The objective of the present work was to investigate a possible mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation, and provide experimental basis for the study of tooth eruption disorder.

Methods: Mouse osteoblast-like (MC3T3-E1) cells were inoculated with a cell density of 70%. According to the grouping experimental design, Western blot and monodansylcadaverine (MDC) detection were conducted after dosing for 24?h. The cells were divided into the following five groups: blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group; 25?µg/mL SN50 group and 50?µg/mL SN50 group.

Results: Western blot analysis revealed that the expression of LC3 protein was present in the blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group and 50?µg/mL SN50 group, with no significant differences among these groups. However, the expression of LC3 protein was significantly lower in the 25?µg/mL SN50 group. MDC detection showed that, in the blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group and 50?µg/mL SN50 group, there was obvious green fluorescence in the cytoplasm of the osteoblasts. However, in the 25?µg/mL SN50 group, it was found that there were significantly fewer green fluorescent particles.

Conclusion: The osteoblast itself had a strong function of autophagy. The appropriate concentration of SN50 in blocking the NF-κB pathway of the osteoblast was associated with the obvious inhibition of autophagy. However, the relationship between NF-κB signaling pathway and autophagy in the process of tooth eruption requires further study.     

MiR-499 induces cardiac differentiation of rat mesenchymal stem cells through wnt/β-catenin signaling pathway

ObjectiveTo test the hypothesis that over-expressing miR-499 in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) induces them to differentiate into cardiomyocyte-like cells through the wnt/β-catenin signaling pathway.MethodsRat BM-MSCs were infected with lentiviral vectors bearing miR-499. The expression of cardiac-specific markers, NKx2.5, GATA4, MEF2C, and cTnI in these cells were examined by rtPCR or Western blot analysis and the activity of the wnt/β-catenin signaling pathway was evaluated by measuring the phosphorylation status of β-catenin.ResultsOver-expression of miR-499 in rat BM-MSCs increased the expression of cardiac-specific genes, such as NKx2.5, GATA4, MEF2C, and cTnI and decreased the ratio of phosphorylated/dephosphorylated β-catenin in the wnt/β-catenin signaling pathway, thus activating the pathway. Knocking down the expression of Dvl, an adaptor molecule in the wnt/β-catenin signaling, partially blocked the role of the miR-499 and decreased those cardiac-specific genes.ConclusionOver-expression of miR-499 in rat BM-MSCs induces them toward cardiac differentiation through the activating the wnt/β-catenin signal pathway.     

3,5-dicaffeoyl‑epi-quinic acid from Atriplex gmelinii enhances the osteoblast differentiation of bone marrow-derived human mesenchymal stromal cells via WnT/BMP signaling and suppresses adipocyte differentiation via AMPK activation

BackgroundImpaired bone formation is one of the reasons behind osteoporosis. Alterations in the patterns of mesenchymal stromal cell differentiation towards adipocytes instead of osteoblasts contribute to osteoporosis progression. Natural anti-osteoporotic agents are effective and safe alternatives for osteoporosis treatment.PurposeIn this context, 3,5-dicaffeoyl‑epi-quinic acid (DCEQA) which is a derivative of chlorogenic acid with reported bioactivities was studied for its osteogenic differentiation enhancing potential in vitro.MethodsAnti-osteoporotic effects of DCEQA were investigated in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) which were induced to differentiate into osteoblasts or adipocytes with or without DCEQA treatment. Changes in the osteogenic and adipogenic markers such as ALP activity and lipid accumulation, respectively, were observed along with differentiation-specific activation of mitogen activated protein kinase (MAPK) pathways.ResultsAt 10 μM concentration, DCEQA increased the proliferation of bone marrow-derived human mesenchymal stromal cells (hBM-MSCs) during osteoblast differentiation. The expression of osteogenic markers ALP, osteocalcin, Runx2, BMP2 and Wnt 10a was upregulated by DCEQA treatment. The ALP activity and extracellular mineralization were also increased. DCEQA elevated the phosphorylation levels of p38 and JNK MAPKs as well as the activation of β-catenin and Smad1/5. DCEQA suppressed the lipid accumulation and downregulated expression of adipogenic markers PPARγ, C/EBPα and SREBP1c in adipo-induced hBM-MSCs. DCEQA also decreased the phosphorylation of p38 and ERK MAPKs and stimulated the activation of AMPK in hBM-MSC adipocytes.ConclusionDCEQA was suggested to enhance osteoblast differentiation via stimulating Wnt/BMP signaling. The adipocyte differentiation inhibitory effect of DCEQA was suggested to arise from its ability to increase AMPK phosphorylation. Overall, DCEQA was shown to possess osteogenesis enhancing and adipogenesis inhibitory properties which might facilitate its use against osteoporotic conditions.     

Semaphorin 3A promotes the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in inflammatory environments by suppressing the Wnt/β-catenin signaling pathway

Journal of Molecular Histology - After periodontal treatment, the local inflammatory environment surrounding periodontal tissues cannot be entirely eliminated. The means by which alveolar bone...     

High glucose enhances TGF-β1 expression in rat bone marrow stem cells via ERK1/2-mediated inhibition of STAT3 signaling

AimsThis study was to investigate the effect of high glucose (HG) on TGF-β1 expression and the underlying mechanisms in bone marrow stem cells.Main methodsRat bone marrow multipotent adult progenitor cells (MAPCs) were cultured in normal (5.5 mM d-glucose) and HG media (25.5 mM d-glucose) for up to 14 days. l-Glucose (20 mM plus 5.5 mM d-glucose) was used as high osmolarity control. TGF-β1 expression was evaluated using quantitative RT-PCR, ELISA, and immunofluorescence staining for its mRNA and protein level in the cells and in the conditioned media. The expression and activation of ERK1/2 and STAT3 were examined in MAPCs cultured in HG media with Western blot.Key findingsMeasurable level of TGF-β1 was detected in the cells cultured in normal media. TGF-β1 expression was substantially increased in MAPCs after 36 h of culture in HG media with over 20-fold increase in the mRNA and 5-fold increase in protein level over control. Interestingly, ERK1/2 phosphorylation was significantly increased in MAPCs cultured in HG media, while in STAT3 (Tyr705), not STAT3 (Ser727), phosphorylation was dramatically decreased. Treatment of cells with the specific MEK1 inhibitor PD98059 or U0126 suppressed ERK1/2 phosphorylation and TGF-β1 expression, and completely restored the level of STAT3 (Tyr705) phosphorylation in MAPCs cultured in HG media. Treatment of the cells with the specific STAT3 phosphorylation inhibitor AG490 significantly blocked STAT3 (Tyr705) phosphorylation and increased TGF-β1 expression without change in ERK1/2 phosphorylation in MPACs.SignificanceHG increased TGF-β1 expression through inhibition of STAT3 (Tyr705) by enhanced ERK1/2 signaling in MAPCs.     

Lipophilic statins antagonistically alter the major epithelial-to-mesenchymal transition signaling pathways in breast cancer stem–like cells via inhibition of the mevalonate pathway

Resistance to therapies, recurrence, and metastasis remain challenging issues for breast cancer patients, particularly for triple-negative and breast cancer stem cells. The activation of the epithelial-to-mesenchymal transition (EMT) plays an indispensable role in the poor prognosis of those types. The accumulating proofs indicated that the mevalonate pathway crucially mediates a poor prognosis. Here, the effects of lipophilic 3-hydroxy-3-methyl-glutaryl-coenzyme A inhibitors, atorvastatin, lovastatin, and simvastatin, were investigated on expression and function of a selected profile of EMT-related genes in breast cancer stem–like cells. A nontoxic dose of statins (5 μM for 4 days) significantly (P < 0.05 and >2-fold change) altered expression of 50 of 71 studied genes with a shared cluster of 37 genes that are coding chief operator of signaling pathways in Hippo, Notch, Wnt, proliferation, invasion, angiogenesis, and cell death. They also significantly decreased the levels of Yap/Taz proteins and shifted the expression of vimentin/E-cadherin in favor of induction of differentiation. Statins significantly chemosensitized the treated cells to doxorubicin and also reduced in vitro migration of the cells. Whereas lovastatin and simvastatin significantly decreased the expression of CD44, atorvastatin drastically increased CD24 and caused more wide-ranging impacts. In summary, the statins hold back the process of EMT by the antagonizing of EMT-promoting pathways. High degree of overlapping findings is supportive of the central role of the mevalonate pathway in cancer stem–like cells, but further studies are required to find the optimized chemical structure for the maximum abrogation of orchestrated EMT pathways.     

Platelet-rich plasma promotes the regeneration of cartilage engineered by mesenchymal stem cells and collagen hydrogel via the TGF-β/SMAD signaling pathway

The tissue engineering technique using mesenchymal stem cells (MSCs) and scaffolds is promising. Transforming growth factor-β1 (TGF-β1) is generally accepted as an chondrogenic agent, but immunorejection and unexpected side effects, such as tumorigenesis and heterogeneity, limit its clinical application. Autogenous platelet-rich plasma (PRP), marked by low immunogenicity, easy accessibility, and low-cost, may be favorable for cartilage regeneration. In our study, the effect of PRP on engineered cartilage constructed by MSCs and collagen hydrogel in vitro and in vivo was investigated and compared with TGF-β1. The results showed that PRP promoted cell proliferation and gene and protein expressions of chondrogenic markers via the TGF-β/SMAD signaling pathway. Meanwhile, it suppressed the expression of collagen type I, a marker of fibrocartilage. Furthermore, PRP accelerated cartilage regeneration on defects with engineered cartilage, advantageous over TGF-β1, as evaluated by histological analysis and immunohistochemical staining. Our work demonstrates that autogenous PRP may substitute TGF-β1 as a potent and reliable chondrogenic inducer for therapy of cartilage defect.     

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.     

Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling

Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca²⁺ signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca²⁺ source is enough for PLC-η1 activation. Furthermore, the IP₃ receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca²⁺ mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca²⁺ mobilization from the ER, and therefore amplifies GPCR-mediated signaling.     

MicroRNA-200c promotes osteogenic differentiation of human bone mesenchymal stem cells through activating the AKT/β-Catenin signaling pathway via downregulating Myd88

During the human bone formation, the event of osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) is vital, and recent evidence has emphasized the important role of microRNAs (miRNAs) in osteogenic differentiation of hBMSCs. This study aims to examine the potential effects of miR-200c in osteogenic differentiation of hBMSCs and understand their underlying mechanisms. HBMSCs were obtained via human bone marrow. During osteogenic induction and differentiation, cells were transfected with different plasmids with the intention of investigating the roles of miR-200c on osteogenic differentiation, calcium salt deposition, alkaline-phosphatase (ALP) activity, mineralized nodule formation, osteocalcin (OCN) content, and proliferation of osteoblasts. Following transfection, dual luciferase reporter gene assay was conducted so as to explore the correlation between miR-200c and Myd88. Moreover, the AKT/β-Catenin signaling pathway was blocked with an AKT/β-Catenin inhibitor, AKTi, to investigate its involvement. The hBMSCs were successfully isolated from human bone marrow. Myd88 was determined as a target gene of miR-200c. Gain and loss-of-function assays confirmed that overexpression of miR-200c, or silencing of Myd88 promoted osteogenic differentiation, increased calcium salt deposition, ALP activity, mineralized nodule formation, and enhanced the proliferation of osteoblasts following osteogenic differentiation of hBMSCs. Meanwhile, the downregulation of miR-200c has been shown to have the opposite effect. Furthermore, these findings showed that the miR-200c overexpression activated the AKT/β-Catenin signaling pathway by targeting Myd88. To sum up, the miR-200c upregulation induces osteogenic differentiation of hBMSCs by activating the AKT/β-Catenin signaling pathway via the inhibition of Myd88, providing a target for treatment of bone repair.     

Establishment of in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells

We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel’s method. We succeeded in establishing an in vitro culture system for evaluating dentin–pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1–3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp–dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.     

Thymosin β4 promotes the migration of endothelial cells without intracellular Ca2+ elevation

Numerous studies have demonstrated the effects of Tβ4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which Tβ4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with Tβ4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that Tβ4 interacts with Ku80, which may operate as a novel receptor for Tβ4 and mediates its intracellular activity. In this paper, we provide evidence that Tβ4 induces cellular processes without changes in the intracellular Ca(2+) concentration. External treatment of HUVECs with Tβ4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (Tβ4(AcSDKPT/4A)) or the actin-binding sequence KLKKTET (Tβ4(KLKKTET/7A)) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by Tβ4 was not associated with the intracellular Ca(2+) elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added Tβ4 induces HUVEC migration via the surface membrane receptors known to generate Ca(2+) influx. Our data confirm the concept that externally added Tβ4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.     

FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-β signaling

Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days’ culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days’ culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-β signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.